【摘要】：越來越多的研究表明，，生精障礙是導(dǎo)致男性不育的重要因素之一。精子生成是一個(gè)復(fù)雜的細(xì)胞發(fā)育與分化的過程，受一個(gè)由多個(gè)基因參與的基因網(wǎng)絡(luò)的高度調(diào)控，這些基因可能既存在性染色體上，也存在常染色體上。分離生精相關(guān)基因是男性不育研究中的一個(gè)重要課題，研究和發(fā)現(xiàn)這些基因，對(duì)于闡明精子發(fā)生的機(jī)制具有重要的生理和病理學(xué)意義。 本課題運(yùn)用了一種發(fā)現(xiàn)人類新基因的新策略“數(shù)據(jù)庫(kù)消減雜交”(Digital Differential Display，DDD)——即通過計(jì)算機(jī)對(duì)不同文庫(kù)進(jìn)行比較來發(fā)現(xiàn)在統(tǒng)計(jì)學(xué)意義上存在明顯轉(zhuǎn)錄差異基因的定量分析方法，結(jié)合RT-PCR、Northern雜交等實(shí)驗(yàn)驗(yàn)證成功克隆了一個(gè)人類睪丸組織特異性表達(dá)的新基因，并探討了其與精子發(fā)生的關(guān)系。本研究分為2個(gè)部分，其主要實(shí)驗(yàn)方法及實(shí)驗(yàn)結(jié)果如下： 第一部分：數(shù)據(jù)庫(kù)消減雜交方法克隆人類睪丸組織特異性表達(dá)新基因TDRG1 1 新基因的電子克隆 美國(guó)NCBI的Unigene數(shù)據(jù)庫(kù)中擁有大量的表達(dá)序列標(biāo)簽(EST)，它們來源于不同組織、不同細(xì)胞、不同病理狀態(tài)下所構(gòu)建的cDNA文庫(kù)，已成為人們尋找新基因的重要標(biāo)志物。Digital DifferentialDisplay(DDD)是一種利用Unigene數(shù)據(jù)庫(kù)中的ESTs進(jìn)行數(shù)種平行材料之間的比較的方法。運(yùn)用該方法，我們進(jìn)行了兩種平行材料之間的比較：挑選9個(gè)人類睪丸組織來源的cDNA序列文庫(kù)作為檢測(cè)子，并將之歸為pool A；將76個(gè)人類其他組織或細(xì)胞株來源的cDNA文庫(kù)序列作為驅(qū)趕子，并將之歸為pool B；通過pool A：pool B之間的比值來計(jì)算倍數(shù)差異，并以≥10倍的差異為篩選標(biāo)準(zhǔn)，經(jīng)數(shù)據(jù)庫(kù)雜交篩選獲得在統(tǒng)計(jì)學(xué)意義上存在差異顯示的克隆重疊群。 在獲得的29個(gè)全新克隆重疊群中，Hs.180197是其中一個(gè)其差異表達(dá)最顯著的克隆重疊群，生物信息學(xué)分析顯示其代表一個(gè)新基因，我們將之作為本課題的研究重點(diǎn)。Hs．180197包括3個(gè)EST，運(yùn)用EST拼接工具進(jìn)行序列匹配、整合，獲得了一個(gè)新的未知序列contig1。將contig1與EST Human數(shù)據(jù)庫(kù)比較，發(fā)現(xiàn)該序列包含另
[Abstract]:More and more studies show that spermatogenic disorder is one of the important factors leading to male infertility. Spermatogenesis is a complex process of cell development and differentiation, which is highly regulated by a network of genes involved in multiple genes, which may exist on both sex and autosomal chromosomes. The isolation of spermatogenic genes is an important subject in the study of male infertility. The study and discovery of these genes have important physiological and pathological significance in elucidating the mechanism of spermatogenesis. In this study, a new strategy for the discovery of new human genes, "Database subtractive Hybridization" (Digital Differential Display,), was used. DDD)-A quantitative analysis of genes with significant transcriptional differences in statistical significance by comparing different libraries with a computer, combined with RT-PCR, A novel gene specifically expressed in human testis was successfully cloned by Northern hybridization and its relationship with spermatogenesis was discussed. This study is divided into two parts. The main experimental methods and results are as follows: part 1: cloning of human testis specific expression gene TDRG1 by database subtractive hybridization Electronic cloning of a new gene Unigene database of NCBI in the United States has a large number of expressed sequence tags (EST), They come from cDNA libraries constructed in different tissues, different cells, different pathological states, . Digital DifferentialDisplay (DDD), an important marker for searching for new genes, is a method to compare several parallel materials using ESTs in Unigene database. Using this method, we compared two kinds of parallel materials: 9 cDNA sequences from human testis were selected as detectors and classified as pool A; The 76 cDNA library sequences from other human tissues or cell lines were used as the banding vectors and classified as pool B; The multiple difference was calculated by the ratio of pool A:pool B, and the screening criterion was the difference of 鈮

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